EP4148376A1 - Procédé d'imagerie d'un objet de mesure à l'aide d'une caméra par microscopie par contraste à interférence différentielle - Google Patents

Procédé d'imagerie d'un objet de mesure à l'aide d'une caméra par microscopie par contraste à interférence différentielle Download PDF

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Publication number
EP4148376A1
EP4148376A1 EP21195744.4A EP21195744A EP4148376A1 EP 4148376 A1 EP4148376 A1 EP 4148376A1 EP 21195744 A EP21195744 A EP 21195744A EP 4148376 A1 EP4148376 A1 EP 4148376A1
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EP
European Patent Office
Prior art keywords
measurement object
camera
measurement
area
polarization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21195744.4A
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German (de)
English (en)
Inventor
Martin Dr. Kraus
Thomas Dr. Engel
Ramona Seliger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics Inc
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Siemens Healthcare Diagnostics Inc
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Filing date
Publication date
Application filed by Siemens Healthcare Diagnostics Inc filed Critical Siemens Healthcare Diagnostics Inc
Priority to EP21195744.4A priority Critical patent/EP4148376A1/fr
Publication of EP4148376A1 publication Critical patent/EP4148376A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B21/00—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant
    • G01B21/10—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant for measuring diameters
    • G01B21/14—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant for measuring diameters internal diameters

Definitions

  • the invention is in the field of automated imaging of a measurement object with a camera in an object area of a microscope using differential interference contrast (DIC).
  • DIC differential interference contrast
  • the basic setting is usually made in the technical contrast of the DIC in such a way that a polarizer and an analyzer are almost exactly crossed, so in image areas without an object there is essentially extinction and the image is dark.
  • phase objects become visible to the eye and also to a camera via the resulting intensity contrast.
  • the object on which the invention is based is therefore the provision of a method for imaging a measurement object with a camera in an object area of a microscope using DIC, which enables improved imaging quality using a camera.
  • an optimized setting should be specified for microscopy with DIC as the imaging method, which is advantageous for the metrological evaluation of DIC images using computer-based methods.
  • Cameras have a linear sensitivity characteristic, so that an adjusted intensity setting in the image is desirable and advantageous compared to observation with the eye, especially with a higher signal level and a linear intensity profile.
  • the invention is consequently based on the finding that the human eye and a camera differ significantly in their detection properties.
  • the eye has a logarithmic characteristic and a very large dynamic range of around 200 dB with black/white and color vision.
  • Cameras on the other hand, have a linear sensitivity curve.
  • Even cameras with so-called Lin/Log sensitivity have linear characteristic curve areas that adjoin one another in sections and have different gradients, which thus simulate the logarithmic curve via the approximation with partial linear areas in sections.
  • the invention further assumes that due to the linear properties of camera sensors in CCD or CMOS technology, it is generally irrelevant at which intensity level within the linearity range of the sensor the measurement signal or the contrasting of the object occurs. In principle, the relative intensity distribution due to the contrasting is retained and can even be optimized in general.
  • the DIC operating point can be shifted to positions that offer advantages for quantitative measurement technology, such as linearity in the medium intensity range of the signal, which also leads to symmetrical intensity changes with positive or negative phase changes in the measurement object. Due to the symmetry newly created by the new setting point, closed path or area integrals result.
  • the numerical value of the integral or the sum of the deviations of the intensity values from the preset value for image areas without an object then assumes the value zero or a value close to zero. This also means that in the case of measurement objects that lie completely in the field of view of the optics or for which a complete overall image can be generated by stitching partial images, this boundary condition for the measurement values can also be achieved.
  • This integration condition can advantageously also be used to evaluate the image quality or the system setting. For example, "closing errors” or a deviation from the ideal value of zero, divided by the number of pixels in the image or the pixels with non-zero information in the image, can be used as a characteristic value for the measurement quality.
  • the basic setting of DIC microscopes is no longer aimed at eliminating the intensity, as was previously the case.
  • the image of areas without an object is therefore not set to dark as usual and, if necessary, the polarization optics with analyzer, polarizer and/or birefringent prism are slightly moved away from the basic setting to give the observer or a camera with some light an orientation in the allow object area.
  • the new adjustment method preferably records amplitude values either in an image or via a scan using the polarization-optical components for DIC adjustment, such as analyzer, polarizer and/or birefringent prism, and then adjusts the components such that the image brightness in object areas without a measurement object corresponds at least approximately to half the amplitude value between maximum and minimum intensity, ie approximately corresponds to a mean amplitude value.
  • the phase angle can be calibrated from the scan curve via the setting angle of the polarization-optical component and the brightness. This calibration can then be used to scale the measured values of the brightness deviations from the mean intensity of object-free image areas.
  • the specific adjustment behavior of the drive for the movement of the polarization-optical component used for the adjustment is advantageously also to be functionally taken into account.
  • One subject of the invention is in particular a method for imaging a measurement object with a camera in an object area of a microscope using differential interference contrast, the microscope comprising an illumination device for illuminating the measurement object, polarization-optical components for realizing the differential interference contrast and the camera for imaging an image of the measurement object in differential interference contrast , whereby the polarization-optical components are set in the basic setting of an adjustment angle such that the image brightness in an area of the object area in which there is no measurement object is at least 20% and at most 80% of the maximum image brightness of a measurement object in the object area, which can be achieved by adjusting the Adjustment angle of the polarization optical components can be achieved.
  • the camera is advantageously a CCD or CMOS camera.
  • the image brightness in the area of the object area in which there is no measurement object is advantageously at least 30% and at most 70% of the maximum image brightness of a measurement object in the object area.
  • the image brightness in the area of the object area in which there is no measurement object is advantageously at least 40% and at most 60%, preferably 50%, of the maximum image brightness of a measurement object in the object area.
  • the polarization-optical components advantageously include at least one polarizer, a polarization analyzer and/or a Wollaston prism, preferably two Wollaston prisms.
  • the chromatic effects of the polarization-optical components for their setting angle are advantageously minimized in the basic setting for a predetermined measurement wavelength range.
  • the lighting device advantageously emits light in a predetermined measurement wavelength range, the measurement wavelength range preferably comprising the wavelengths 455 nm, 490 nm and/or 680 nm.
  • the image brightness in the area of the object area in which there is no measurement object advantageously relates to a mean wavelength of the measurement wavelength range.
  • the mean wavelength corresponds to a weighted average of wavelengths of the measurement wavelength range, weightings preferably being selected which correspond to the importance of the respective measurement wavelength for the imaging to be undertaken.
  • the camera is advantageously designed as a black-and-white camera and/or a color camera.
  • a color camera is designed in the Bayer design or as a multi-chip color camera, or instead of a color camera, separate color cameras are provided for different color channels.
  • the measurement object advantageously comprises a biological cell, preferably a blood cell, urine sediment and/or a medical preparation, the measurement object preferably being taken from a human or an animal and/or being provided before the method is carried out.
  • an automatic analysis device comprising a microscope for imaging a measurement object with a camera in an object area of the microscope using differential interference contrast, the microscope comprising an illumination device for illuminating the measurement object, polarization-optical components for realizing the differential interference contrast and the camera for imaging an image of the measurement object in differential interference contrast, the automatic analysis device further comprising a programmable control device, wherein the control device is configured such that the automatic analysis device can carry out a method according to the invention.
  • the automatic analyzer comprises an automatic hematology analyzer or is designed as an automatic hematology analyzer.
  • Another object of the invention is the use of a method according to the invention in an automatic analysis device, preferably in an automatic analysis device according to the invention.
  • FIG 1 The course shown of the intensity incident on the detector in DIC operation as a function of the setting of the polarization-optical component for setting the DIC contrast at 445 nm (blue) central measuring wavelength ( ⁇ ) was measured with a Leica DMi 8 microscope, adjustment range +/- 20,000 increments, Increment per step 500 increments, measured.
  • the position of the slider ("slider position" A) is plotted in arbitrary units on the X-axis against the background intensity ("background intensity" B).
  • FIG 2 and 3 show corresponding curves as in FIG 1 at 490 nm (D) and 680 nm (E) central measuring wavelength.
  • the curves show the minimum for the classic setting of the DIC contrast at about step 40.
  • the curves are very flat in the area around step 40, with little slope and are therefore practically asymptotic to the x-axis of the plots.
  • the slight asymmetries from the right to the left part of the curve are probably due to technical reasons and are based on the manufacturing quality of the entire optical system.
  • the curves show the greatest slope, as this is the middle area between the min and max reversal points of the sinusoidal curve.
  • the DIC operating point can thus be shifted to positions that offer advantages for quantitative measurement technology, such as linearity in the medium intensity range of the signals.
  • this also leads to symmetrical changes in intensity with positive or negative phase changes in the measurement object.
  • This newly created symmetry due to the new setting point allows further closed path or area integrals, in which the numerical value of the integral or the sum of the deviations of the intensity values from the preset value for image areas without an object then assumes the value zero or a value close to zero .
  • This boundary condition for the measured values can also be achieved for measurement objects that lie completely in the field of view of the optics or for which a complete overall image can be generated by stitching partial images.
  • This integration condition can advantageously also be used to evaluate the image quality or the system setting, e.g. "closing error” or the deviation from the ideal value of zero divided by the number of pixels in the image or pixels with non-zero information in the image as characteristic value for the measurement quality is used.
  • FIG 4 shows the three representations of the 1, 2 and 3 united in one picture.
  • the three curves shown are similar in trough and share a common trough near step 40 . Starting from this low point, the curves increasingly diverge in the relative phase position of the sinusoidal curve. Due to the higher material dispersion at 455 nm (blue) compared to green (490 nm) and red (680 nm), the curve for blue is steepest or has the shortest period and the curve for red has the longest period.
  • FIG 5 shows the curves for better comparability FIG 4 in a normalized representation in terms of Y-axis plotted background intensity ("Background Intensity" B).
  • a setting of the slider is advantageously chosen such that for the shortest wavelength and the longest wavelength the background intensity B is still as centered as possible in the linear range.
  • the measurement wavelengths are advantageously 455 nm, 490 nm and 680 nm, for example.
  • spectral bands are advantageously used for the measurement.
  • a setting (increment, as 500 times smaller than a step) is preferably selected in which the mean intensities for the colors considered are centered as far as possible in their range around the mean value or the range between the points of the wavelengths is minimized as far as possible .
  • the outer wavelengths preferably satisfy an additional boundary condition, such as not falling below 20% and not exceeding 80%. Alternatively, it is particularly preferred, for example, to not fall below 30% and not to exceed 70%.
  • the positions (increments) of the settings are preferably determined as an alternative criterion for the setting for the respective measurement wavelengths or measurement wavelength ranges.
  • the mean value of these two increment values then gives the setting position for the respective wavelength.
  • it is then advantageously sufficient eg in the basic setting of the device when switching on or when checking or qualifying the function, only to determine the position of the minimum and from there to set a fixed value of an offset in increments, for example in a device qualification or device acceptance test.
  • phase adjustment per increment of the drive can also be determined during acceptance, so that other, optimized settings can also be made later without renewed referencing measurements.
  • a permitted phase range for a linear contrasting behavior and/or a substantially symmetrical contrasting behavior is known from the referencing measurements, e.g. in the form that the phase is e.g. in the range 20 to 70 degrees or 30 to 60 degrees for all wavelengths used in the measurement should.
  • This phase criterion would then be equivalent to an intensity criterion.
  • control behavior or limit range between wavelength ranges are interpolated. This has the advantage that the characterization effort for the system can be limited for referencing measurements.
  • chromatically corrected splitting prisms e.g. in the sense of low order or zero order retarder plates, by either minimizing the thickness of the plates and/or the splitting primate, such as a Wollaston or Nomarski prism, from more than two birefringent pieces of material arranged to minimize the chromatic effects for the tilt angle of the polarizing element over a predetermined range of wavelengths.
  • the splitting primate such as a Wollaston or Nomarski prism
  • a combination of splitting prism and retardation plate is advantageous for chromatic correction.
  • a half-wave plate also referred to as a lambda/2 plate, preferably in a low-order or higher-order design, with the appropriate orientation of the axes is inserted into the beam path, e.g. in front of the analyzer.
  • a combination of a color camera and an associated color evaluation is advantageously used instead of a black/white camera. This has the advantage that more use can be made of the chromatic properties of the DIC system.
  • the color camera is advantageously implemented in the Bayer design or, in order to retain an optimized resolution, designed as a multi-chip color camera.
  • the camera comprises at least two separate cameras for different color channels, with exactly one or at least one camera preferably being provided for each color channel.
  • the example in figure 5 normalization shown for the respective color channel, for example, by means of the intensity setting of the light source and/or via the camera setting for, for example, the gain and/or the exposure time.
  • a scaling factor predetermined by means of device referencing is advantageously used as an alternative for the color channels.
  • the color channels are advantageously recorded serially with a black/white camera and specific operating parameters are used for at least one of a plurality of color channels, preferably for each of the color channels.

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  • Life Sciences & Earth Sciences (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
EP21195744.4A 2021-09-09 2021-09-09 Procédé d'imagerie d'un objet de mesure à l'aide d'une caméra par microscopie par contraste à interférence différentielle Withdrawn EP4148376A1 (fr)

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EP21195744.4A EP4148376A1 (fr) 2021-09-09 2021-09-09 Procédé d'imagerie d'un objet de mesure à l'aide d'une caméra par microscopie par contraste à interférence différentielle

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EP21195744.4A EP4148376A1 (fr) 2021-09-09 2021-09-09 Procédé d'imagerie d'un objet de mesure à l'aide d'une caméra par microscopie par contraste à interférence différentielle

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4412246A (en) * 1981-07-13 1983-10-25 Hamamatsu Systems, Inc. Method of adjusting a video microscope system incorporating polarization or interference optics for optimum imaging conditions
US20030161038A1 (en) * 2000-06-14 2003-08-28 Helmut Tobben Microscope and method for measuring surface topography in a quantitative and optical manner
US20210217190A1 (en) * 2018-05-30 2021-07-15 Siemens Healthcare Diagnostics Inc. Analyzer for three-dimensionally analyzing a medical sample by means of a light field camera

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4412246A (en) * 1981-07-13 1983-10-25 Hamamatsu Systems, Inc. Method of adjusting a video microscope system incorporating polarization or interference optics for optimum imaging conditions
US20030161038A1 (en) * 2000-06-14 2003-08-28 Helmut Tobben Microscope and method for measuring surface topography in a quantitative and optical manner
US20210217190A1 (en) * 2018-05-30 2021-07-15 Siemens Healthcare Diagnostics Inc. Analyzer for three-dimensionally analyzing a medical sample by means of a light field camera

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HOLZWARTH G M ET AL: "POLARIZATION-MODULATED DIFFERENTIAL-INTERFERENCE CONTRAST MICROSCOPY WITH A VARIABLE RETARDER", APPLIED OPTICS, OPTICAL SOCIETY OF AMERICA, WASHINGTON, DC, US, vol. 39, no. 34, 1 December 2000 (2000-12-01), pages 6288 - 6294, XP001015096, ISSN: 0003-6935, DOI: 10.1364/AO.39.006288 *

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